Contrasting gut bacteriomes unveiled between wild Antheraea assamensis Helfer (Lepidoptera: Saturniidae) and domesticated Bombyx mori L. (Lepidoptera: Bombycidae) silkworms.

BACKGROUND: Insect gut microbiomes play a fundamental role in various aspects of insect physiology, including digestion, nutrient metabolism, detoxification, immunity, growth and development. The wild Muga silkworm, Antheraea assamensis Helfer holds significant economic importance, as it produces golden silk. METHODS AND RESULTS: In the current investigation, we deciphered its intricate gut bacteriome through high-throughput 16S rRNA amplicon sequencing. Further, to understand bacterial community dynamics among silkworms raised under outdoor environmental conditions, we compared its gut bacteriomes with those of the domesticated mulberry silkworm, Bombyx mori L. Most abundant bacterial phyla identified in the gut of A. assamensis were Proteobacteria (78.1%), Bacteroidetes (8.0%) and Firmicutes (6.6%), whereas the most-abundant phyla in B. mori were Firmicutes (49-86%) and Actinobacteria (10-36%). Further, Gammaproteobacteria (57.1%), Alphaproteobacteria (10.47%) and Betaproteobacteria (8.28%) were the dominant bacterial classes found in the gut of A. assamensis. The predominant bacterial families in A. assamensis gut were Enterobacteriaceae (27.7%), Comamonadaceae (9.13%), Pseudomonadaceae (9.08%) Flavobacteriaceae (7.59%) Moraxellaceae (7.38%) Alteromonadaceae (6.8%) and Enterococcaceae (4.46%). In B. mori, the most-abundant bacterial families were Peptostreptococcaceae, Enterococcaceae, Lactobacillaceae and Bifidobacteriaceae, though all showed great variability among the samples. The core gut bacteriome of A. assamensis consisted of Pseudomonas, Acinetobacter, Variovorax, Myroides, Alteromonas, Enterobacter, Enterococcus, Sphingomonas, Brevundimonas, Oleispira, Comamonas, Oleibacter Vagococcus, Aminobacter, Marinobacter, Cupriavidus, Aeromonas, and Bacillus. Comparative gut bacteriome analysis revealed a more complex gut bacterial diversity in wild A. assamensis silkworms than in domesticated B. mori silkworms, which contained a relatively simple gut bacteriome as estimated by OTU richness. Predictive functional profiling of the gut bacteriome suggested that gut bacteria in A. assamensis were associated with a wide range of physiological, nutritional, and metabolic functions, including biodegradation of xenobiotics, lipid, amino acid, carbohydrate metabolism, and biosynthesis of secondary metabolites and amino acids. CONCLUSIONS: These results showed great differences in the composition and diversity of gut bacteria between the two silkworm species. Both insect species harbored core bacterial taxa commonly found in insects, but the relative abundance and composition of these taxa varied markedly.

Genome sequencing and assembly of Indian golden silkmoth, Antheraea assamensis Helfer (Saturniidae, Lepidoptera).

Muga silkworm (Antheraea assamensis), one of the economically important wild silkmoths, is unique among saturniid silkmoths. It is confined to the North-eastern part of India. Muga silk has the highest value among the other silks. Unlike other silkmoths, A. assamensis has a low chromosome number (n = 15), and ZZ/ZO sex chromosome system. Here, we report the first high-quality draft genome of A. assamensis, assembled by employing the Illumina and PacBio sequencing platforms. The assembled genome of A. assamensis is 501.18 Mb long, with 2697 scaffolds and an N50 of 683.23 Kb. The genome encompasses 18,385 protein-coding genes, 86.29% of which were functionally annotated. Phylogenetic analysis of A. assamensis revealed its divergence from other Antheraea species approximately 28.7 million years ago. Moreover, an investigation into detoxification-related gene families, CYP450, GST, and ABC-transporter, revealed a significant expansion in A. assamensis as compared to the Bombyx mori. This expansion is comparable to Spodoptera litura, suggesting adaptive responses linked to the polyphagous behavior observed in these insects. This study provides valuable insights into the molecular basis of evolutionary divergence and adaptations in muga silkmoth. The genome assembly reported in this study will significantly help in the functional genomics studies on A. assamensis and other Antheraea species along with comparative genomics analyses of Bombycoidea insects.

Comparative seasonal analysis of Eri silkworm (Samia ricini Donovan) gut composition: implications for lignocellulose degradation.

Conversion of biomass such as lignocelluloses to an alternative energy source can contribute to sustainable development. Recently, biomass-degrading enzymes are reported to be common resources in insect-microbe interacting systems. Northeast India harbors ample sericigenous insect resources which are exploited for their silk products. Samia ricini Donovan is an economically important poly-phytophagous silkmoth capable of digesting foliage from different plant species, suggesting the versatility of a robust gut system. Here, a gut bacterial profile was determined by 16S rRNA gene characterization across the holometabolous life cycle during the summer and winter seasons, revealing 3 phyla, 13 families, and 22 genera. Comparative analysis among the seasonal gut isolates revealed a high diversity in summer, predominated by the genus Bacillus due to its high occurrence in all developmental stages. Shannon's diversity index demonstrated the second and fourth instars of summer as well as the fifth instar of winter to be relatively better developmental stages for gut bacteria assembly. Bacterial community shifts in concert to host developmental changes were found to be apparent between early instars and late instars in summer, which differed from those of winter. Forty-three and twenty-nine gut bacterial isolates were found to be cellulolytic and xylanolytic enzyme producers, respectively. The present results illustrate the gut microbiota of S. ricini over the seasons and support the holometabolous life cycle effect as the most likely factor shaping the gut bacterial microbiota. These findings may provide leads for the development of new cleaner and environmentally friendly lignocellulose-degrading enzymes.

De novogenomic analysis ofEnterobacter asburiaeEBRJ12, a plant growth-promoting rhizobacteria isolated from the rhizosphere of Phaseolus vulgarisL.

AIM: Environmental stresses such as water deficit induced stress are one of the major limiting factors in crop production. However, some plant growth-promoting rhizobacteria (PGPR) can promote plant growth in such adverse condition. Therefore, the objective was to isolate rhizospheric bacteria from Phaseolus vulgaris L. growing in a drought-affected soil and to analyze its plant growth promoting (PGP) efficacy to black gram (Vigna mungo L.) and Bhut jolokia (Capsicum chinense Jacq.). Whole-genome sequencing of the potential bacteria was targeted to analyze the genetic potential of the isolate as a plant growth-promoting agent. METHODS AND RESULTS: The isolate Enterobacter asburiae EBRJ12 was selected based on its PGP efficacy, which significantly improved plant growth and development. The genomic analysis revealed the presence of one circular chromosome of size 4.8 Mb containing 16 genes for osmotic stress regulation including osmotically inducible protein osmY, outer membrane protein A precursor ompA, aquaporin Z, and an operon for osmoprotectant ABC transporter yehZYXW. Moreover, the genome has a complete genetic cluster for biosynthesis of siderophore Enterobactin and siderophore Aerobactin.The PGP effects were verified with black gram and Bhut jolokia in pot experiments. The isolate significantly increased the shoot length by 35.0% and root length by 58.0% of black gram, while 41.0% and 57.0% of elevation in shoot and root length were observed in Bhut jolokia compared to non-inoculated plants. CONCLUSIONS: The EBRJ12 has PGP features that could improve the growth in host plants, and the genomic characterization revealed the presence of genetic potential for plant growth promotion.

Phylogenetic affiliation and antimicrobial effects of endophytic actinobacteria associated with medicinal plants: prevalence of polyketide synthase type II in antimicrobial strains.

The most diverse and versatile endophytic actinobacteria are relatively unexplored potential sources of bioactive metabolites useful for different medical, agricultural, and other commercial applications. Their diversity in symbiotic association with traditionally utilized medicinal plants of northeast India is scantly available. The present investigation assessed the genetic diversity of endophytic actinobacteria (n = 120) distributed around the root, stem, and leaf tissues of six selected medicinal plants (Emblica officinalis, Terminalia chebula, T. arjuna, Murraya koenigii, Rauwolfia serpentina, and Azadirachta indica) from three different protected areas of evergreen forest-the Gibbon Wildlife Sanctuary (GWS), the Kaziranga National Park (KNP), and the North East Ecological Park (NEEP) of Assam, India. The samples were collected in two seasons (summer and winter). The overall phylogenetic analysis showed significant genetic diversity with 18 distinct genera belonging to 12 families. Overall, the occurrence of Streptomyces genus was predominant across all three sampling sites (76.66%), in both the sampling season (summer and winter). Shannon's and Simpson's diversity estimates showed their presence at A. indica (1.496, 0.778), R. serpentina (1.470, 0.858), and E. officinalis (0.975, 0.353). Among the site sampled, GWS had the most diverse community of actinobacteria (Shannon = 0.86 and Simpson = 0.557). The isolates were antagonistically more active against the investigated plant pathogenic bacteria than fungal pathogens. Further analysis revealed the prevalence of polyketide synthase genes (PKS) type II (84%) and PKS type I (16%) in the genome of the antimicrobial isolates. The overall findings confirmed the presence of biosynthetically active diverse actinobacterial members in the selected medicinal plants which offer potential opportunities towards the exploration of biologically active compounds.

Rhizospheric Bacterial Community of Endemic Rhododendron arboreum Sm. Ssp. delavayi along Eastern Himalayan Slope in Tawang.

Information on rhizosphere microbiome of endemic plants from high mountain ecosystems against those of cultivated plantations is inadequate. Comparative bacterial profiles of endemic medicinal plant Rhododendron arboreum Sm. subsp. delavayi rhizosphere pertaining to four altitudinal zonation Pankang Thang (PTSO), Nagula, Y-junction and Bum La (Indo-China border; in triplicates each) along cold adapted Eastern slope of Himalayan Tawang region, India is described here. Significant differences in DGGE profile between below ground bulk vs. rhizospheric community profile associated with the plant was identified. Tagged 16S amplicon sequencing from PTSO (3912 m) to Bum La (4509 m), revealed that soil pH, total nitrogen (TN), organic matter (OM) significantly influenced the underlying bacterial community structure at different altitudes. The relative abundance of Acidobacteria was inversely related to pH, as opposed to TN which was positively correlated to Acidobacteria and Proteobacteria abundance. TN was also the significant predictor for less abundant taxonomic groups Chloroflexi, Gemmatimonadetes, and Nitrospirae. Bum La soil harbored less bacterial diversity compared to other sites at lower altitudes. The most abundant phyla at 3% genetic difference were Acidobacteria, Actinobacteria, and Proteobacteria amongst others. Analysis of similarity indicated greater similarity within lower altitudinal than higher altitudinal group (ANOSIM, R = 0.287, p = 0.02). Constraining the ordination with the edaphic factor explained 83.13% of variation. Unique phylotypes of Bradyrhizobium and uncultured Rhizobiales were found in significant proportions at the four regions. With over 1% relative abundance Actinobacteria (42.6%), Acidobacteria (24.02%), Proteobacteria (16.00%), AD3 (9.23%), WPS-2 (5.1%), and Chloroflexi (1.48%) dominated the core microbiome.

Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds.

Community profiling of culturable fluorescent pseudomonads in the rhizosphere of green gram (Vigna radiata L.).

Study on microbial diversity in the unexplored rhizosphere is important to understand their community structure, biology and ecological interaction with the host plant. This research assessed the genetic and functional diversity of fluorescent pseudomonads [FP] in the green gram rhizophere. One hundred and twenty types of morphologically distinct fluorescent pseudomonads were isolated during vegetative as well as reproductive growth phase of green gram. Rep PCR, ARDRA and RISA revealed two distinct clusters in each case at 75, 61 and 70% similarity coefficient index respectively. 16S rRNA partial sequencing analysis of 85 distantly related fluorescent pseudomonads depicted Pseudomonas aeruginosa as the dominant group. Out of 120 isolates, 23 (19%) showed antagonistic activity towards phytopathogenic fungi. These bacterial isolates showed varied production of salicylic acid, HCN and chitinase, 2, 4-diacetylphloroglucinol (DAPG), phenazine-1-carboxylic acid (PCA) and pyoluteorin (PLT). Production efficiency of inherent level of plant growth promoting (PGP) traits among the 120 isolates demonstrated that 10 (8%) solubilised inorganic phosphates, 25 (20%) produced indoles and 5 (4%) retained ACC deaminase activity. Pseudomonas aeruginosa GGRJ21 showed the highest production of all antagonistic and plant growth promoting (PGP) traits. In a greenhouse experiment, GGRJ21 suppressed root rot disease of green gram by 28-93% (p = 0.05). Consistent up regulation of three important stress responsive genes, i.e., acdS, KatA and gbsA and elevated production efficiency of different PGP traits could promote GGRJ21 as a potent plant growth regulator.

Psychrotolerant antifungal Streptomyces isolated from Tawang, India and the shift in chitinase gene family.

A total of 210 Streptomyces were isolated from the soil samples of Tawang, India where temperature varied from 5 °C during daytime to -2 °C during the night. Based on antifungal activity, a total of 33 strains, putatively Streptomyces spp., were selected. Optimal growth temperature for the 33 strains was 16 °C, with growth occurring down to 6 °C but not above 30 °C. Phylogenetic analysis based on 16S rDNA sequences revealed the taxonomic affiliation of the 33 strains as species of Streptomyces. To examine the relatedness of the chitinase genes from six strong antifungal Streptomyces strains, a phylogenetic tree was constructed using the catalytic domain nucleotide sequences and resulted in seven distinct monophyletic groups. A quantitative PCR study for chitinase expressing ability revealed that of the six antifungal strains tested, the strain Streptomyces roseochromogenus TSR12 was the most active producer of family 18 chitinase genes. Streptomyces strains with enhanced inhibitory potential usually encode a family 19 chitinase gene; however, our present study did not show expression of this family in the six strains tested.

Bioprospecting Micromonospora from Kaziranga National Park of India and their anti-infective potential.

Large number of strains was isolated from soils of Kaziranga National Park of North-East India using selective isolation procedure. They were assigned to the genus Micromonospora on the basis of their typical colonial and pigmentation features. The taxonomic identities of the isolates were confirmed on the basis of their molecular characters (16SrDNA). A total of one hundred Micromonospora strains were isolated during the present investigation. The diagnostic cell wall sugar and amino acids were determined from these Micromonospora strains. After preliminary screening most of the isolates exhibited excellent anti-infective activity against human bacterial pathogens Staphylococcus aureas, Bacillus subtilis, Proteus vulgaris, Echerichia coli, Pseudomonas aeroginosa and fungal pathogens Aspergillus niger, Fusarium oxysporum and Candida albicans. Among these isolates one strain designated as HK-10 showed promising activity against human pathogens S. aureas, B. subtilis, P. vulgaris and P. aeroginosa.

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (Vigna radiata L.) rhizosphere.

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099(T)), Pseudomonas aureofaciens (NCIM2026(T)), and Pseudomonas aeruginosa (MTCC2582(T)). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH(4))(2)SO(4) precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn(2+), Cu(2+), and Ni(2+) ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.